Department of Applied Chemistry, Graduate School of Science and Engineering, University of Toyama, Gofuku 3190, Toyama 930-8555, Japan.
Colloids Surf B Biointerfaces. 2010 Jul 1;78(2):155-62. doi: 10.1016/j.colsurfb.2010.02.022. Epub 2010 Feb 25.
A novel disulfide, which carried a specific inhibitor for beta-secretase (KMI360) at both ends, was prepared by the coupling of 11,11'-dithiobisundecanoic acid (DTUA) with the inhibitor. The compound obtained (DTUA-KMI360) formed a self-assembled monolayer (SAM) on a gold electrode as proven by cyclic voltammetry (CV) using hydroquinone as a probe. Furthermore, DTUA-KMI360 could be accumulated as a SAM on a gold colloid deposited on a glass plate (Au colloid-glass chip) as proven by both the red-shift and the increase in absorbance of the gold colloid corresponding to localized surface plasmon resonance (LSPR). When the SAM-modified Au colloid-glass chip was immersed in a solution of aspartyl proteases, pepsin and beta-secretase, the absorbance of the chip at 550nm corresponding to LSPR of the gold colloid further increased and was slightly red-shifted, whereas coexistence of a free inhibitor obstructed these phenomena. Adsorption of the enzymes was promoted by the incorporation of a zwitterionic group into the SAM, while non-specific adsorption to the mixed SAM was significantly reduced. The optimal ratio of omega-zwitterionic alkanethiol, 3-[(6-mercaptohexyl)-N,N-dimethylamino]propane-1-sulfonic acid (C(6)-SPB), and DTUA-KMI360 in the SAM for the binding of enzymes was found to be DTUA-KMI360:C(6)-SPB=1:11 using polarization modulation infrared reflection absorption spectroscopy (PM-IR-RAS). From increasing profiles of absorbance of the Au colloid-glass chip, the association constant (K(assoc)) for pepsin with the inhibitor on the SAM was determined, whereas that for beta-secretase could not be due to the strong binding of the enzyme to the inhibitor, resulting in the absence of the dissociation process. The results suggested that the SAM of the enzyme inhibitor can be used for both investigation of enzymes and removal of target enzymes from biological fluids.
一种新型二硫键，在两端都携带了β-分泌酶（KMI360）的特定抑制剂，是通过 11,11'-二硫代十一烷酸（DTUA）与抑制剂的偶联制备的。通过使用氢醌作为探针的循环伏安法（CV）证明，所得化合物（DTUA-KMI360）在金电极上形成了自组装单层（SAM）。此外，DTUA-KMI360 可以作为 SAM 被聚集在沉积在玻璃片上的金胶上（Au 胶体-玻璃芯片），这可以通过金胶的局部表面等离子体共振（LSPR）的红移和吸光度增加来证明。当 SAM 修饰的 Au 胶体-玻璃芯片被浸入天冬氨酸蛋白酶、胃蛋白酶和β-分泌酶的溶液中时，对应于金胶的 LSPR 的芯片在 550nm 处的吸光度进一步增加并略有红移，而游离抑制剂的共存则阻碍了这些现象。将两性离子基团引入 SAM 中促进了酶的吸附，同时显著减少了非特异性吸附到混合 SAM。使用偏振调制红外反射吸收光谱（PM-IR-RAS）发现，在 SAM 中，ω-两性离子烷硫醇 3-[(6-巯基己基)-N,N-二甲基氨基]丙烷-1-磺酸（C（6）-SPB）和 DTUA-KMI360 的最佳比例为 1:11，用于酶的结合。从 Au 胶体-玻璃芯片的吸光度增加曲线确定了抑制剂与 SAM 上胃蛋白酶的结合常数（K（assoc）），而对于β-分泌酶则不能，因为酶与抑制剂的强结合导致没有解离过程。结果表明，酶抑制剂的 SAM 可用于研究酶并从生物流体中去除靶酶。
